Mediator For Test Sensor

ABSTRACT

A method of forming a 3-phenylimino-3H-phenothiazine or a 3-phenylimino-3H-phenoxazine mediator includes providing a first reactant including phenothiazine or phenoxazine, providing a first solvent, providing a second reactant and providing a second solvent. The first reactant, first solvent, second reactant and second solvent are combined to form a reactants solution. Sodium persulfate is added to the reactants solution to couple the first and second reactants resulting in a reaction solution including the 3-phenylimino-3H-phenothiazine or the 3-phenylimino-3H-phenoxazine mediator.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation application of application Ser. No.13/717,024 filed Dec. 17, 2012, which has been allowed; application Ser.No. 13/717,024 is a continuation application of application Ser. No.13/207,855, filed on Aug. 11, 2011, which issued as U.S. Pat. No.8,357,797 on Jan. 22, 2013; application Ser. No. 13/207,855 is adivisional application of application Ser. No. 12/316,115 filed on Dec.9, 2008, which issued as U.S. Pat. No. 8,022,204 on Sep. 20, 2011,application Ser. No. 12/316,115 claims benefit of ProvisionalApplication No. 61/007,178 filed on Dec. 10, 2007, all of which areincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention generally relates to a method forming a mediator.More specifically, the present invention generally relates to a methodof forming a mediator to be used in an electrochemical test sensor thatis adapted to assist in determining information related to an analyte.

BACKGROUND OF THE INVENTION

The quantitative determination of analytes in body fluids is of greatimportance in the diagnoses and maintenance of certain physicalconditions. For example, lactate, cholesterol and bilirubin should bemonitored in certain individuals. In particular, it is important thatindividuals with diabetes frequently check the glucose level in theirbody fluids to regulate the glucose intake in their diets. The resultsof such tests can be used to determine what, if any, insulin or othermedication needs to be administered. In one type of blood-glucosetesting system, test sensors are used to test a sample of blood.

A test sensor contains biosensing or reagent material that reacts with,for example, blood glucose. One type of electrochemical test sensor is amultilayer test sensor that includes a base or substrate and a lid.Another type of electrochemical test sensor includes a base, a spacerand a lid. Existing electrochemical test sensors include at least twoelectrodes in the form of an electrode pattern. A potential is appliedacross these electrodes and a current is measured at the workingelectrode. The current measurement is directly proportional to the sizeof the working electrode.

Electrochemical test sensors are based on enzyme-catalyzed chemicalreactions involving the analyte of interest. In the case of glucosemonitoring, the relevant chemical reaction is the oxidation of glucoseto gluconolactone or its corresponding acid. This oxidation is catalyzedby a variety of enzymes, some of which may use coenzymes such asnicotinamide adenine dinucleotide (phosphate) (NAD(P)), while others mayuse coenzymes such as flavin adenine dinucleotide (FAD) orpyrroloquinolinequinone (PQQ).

In test-sensor applications, the redox equivalents generated in thecourse of the oxidation of glucose are transported to the surface of anelectrode, whereby an electrical signal is generated. The magnitude ofthe electrical signal is then correlated with glucose concentration. Thetransfer of redox equivalents from the site of chemical reaction in theenzyme to the surface of the electrode is accomplished using electrontransfer mediators.

Many mediators such as, for example, ferricyanide have a high backgroundcurrent such that the signal-to-noise ratio when formulated in a glucosetest sensor is low. Typically, a low signal-to-noise ratio results in ahigher assay imprecision, particularly at lower glucose levels and highhematocrit sample levels. With quicker sample tests (e.g., test timesless than 10 seconds), it may be difficult to burn off the highbackground current in the time allocated to perform the test. Because ofthe quicker sample test times, this necessitates that the activeingredients interact rapidly when sample is applied to give a rapidresponse.

Therefore, it would be desirable to form a mediator that has a lowbackground current, while still having other desirable attributes of amediator including stability.

SUMMARY OF THE INVENTION

A method of forming a 3-phenylimino-3H-phenothiazine mediator includesproviding a first reactant including phenothiazine. A first solvent isprovided in which the phenothiazine has a desired solubility therein. Asecond reactant is provided to assist in forming the3-phenylimino-3H-phenothiazine mediator. A second solvent is provided inwhich the second reactant has a desired solubility therein. The firstreactant, first solvent, second reactant and second solvent are combinedto form a reactants solution. Sodium persulfate is added to thereactants solution to couple the first and second reactants resulting ina reaction solution including the 3-phenylimino-3H-phenothiazinemediator. After adding the sodium persulfate, the reaction solution isfurther processed to include the 3-phenylimino-3H-phenothiazine mediatorso as to isolate the 3-phenylimino-3H-phenothiazine mediator.

A method of forming a 3-phenylimino-3H-phenoxazine mediator includesproviding a first reactant including phenoxazine. A first solvent isprovided in which the phenoxazine has a desired solubility therein. Asecond reactant is provided to assist in forming the3-phenylimino-3H-phenoxazine mediator. A second solvent is provided inwhich the second reactant has a desired solubility therein. The firstreactant, first solvent, second reactant and second solvent are combinedto form a reactants solution. Sodium persulfate is added to thereactants solution to couple the first and second reactants resulting ina reaction solution including the 3-phenylimino-3H-phenoxazine mediator.After adding the sodium persulfate, the reaction solution is furtherprocessed to include the 3-phenylimino-3H-phenoxazine mediator so as toisolate the 3-phenylimino-3H-phenoxazine mediator.

A method of forming and stabilizing a 3-phenylimino-3H-phenothiazinemediator or 3-phenylimino-3H-phenoxazine mediator includes providing afirst reactant including phenothiazine or phenoxazine. A first solventis provided in which the phenothiazine or the phenoxazine has a desiredsolubility therein. A second reactant is provided to assist in formingthe 3-phenylimino-3H-phenothiazine mediator or the3-phenylimino-3H-phenothiazine mediator. A second solvent is provided inwhich the second reactant has a desired solubility therein. The firstreactant, first solvent, second reactant and second solvent are combinedto form a reactants solution. A coupling agent is added to the reactantssolution to couple the first and second reactants resulting in areaction solution including the 3-phenylimino-3H-phenothiazine mediatoror the 3-phenylimino-3H-phenoxazine mediator. After adding the couplingagent, the reaction solution is further processed to include the3-phenylimino-3H-phenothiazine mediator or the3-phenylimino-3H-phenoxazine mediator so as to isolate the3-phenylimino-3H-phenothiazine mediator or the3-phenylimino-3H-phenoxazine mediator. The3-phenylimino-3H-phenothiazine mediator or the3-phenylimino-3H-phenoxazine mediator is stabilized to a pH of fromabout 5 to about 8.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is a test sensor according to one embodiment.

FIG. 1 b is a side view of the test sensor of FIG. 1 a.

FIG. 2 is a plot of background current versus various lots of inventiveand comparative mediators.

FIG. 3 a is a plot of background current using several neutralization orbuffering processes and some processes without neutralization orbuffering.

FIG. 3 b is a plot of change in background current between a baselineand the background current measured in FIG. 3 a.

DETAILED DESCRIPTION OF ILLUSTRATED EMBODIMENTS

In one process, the present invention is directed to an improved methodof producing a low background current 3-phenylimino-3H-phenothiazinemediator or 3-phenyliminio-3H-phenoxazine mediator. In another process,the present invention is directed to an improved method of stabilizing alow background current 3-phenylimino-3H-phenothiazine mediator or3-phenyliminio-3H-phenoxazine mediator. The3-phenylimino-3H-phenothiazine mediators or3-phenyliminio-3H-phenoxazine mediators are useful mediators forelectrochemical test sensors and in one example are useful in theelectrochemical regeneration (oxidation) of NADH.

Mediators to be formed in the present invention include phenothiazineshaving the formula

and phenoxazines having the formula

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are the same or differentand are independently selected from the group consisting of hydrogen,alkyl, alkenyl, alkynyl, aryl, heteroaryl, cyclic, heterocyclic, halo,haloalkyl, carboxy, carboxyalkyl, alkoxycarbonyl, aryloxycarbonyl,aromatic keto, aliphatic keto, alkoxy, aryloxy, nitro, dialkylamino,aminoalkyl, sulfo, dihydroxyboron, and combinations thereof. It iscontemplated that isomers of the same may also be formed.

The 3-phenylimino-3H-phenothiazine mediator or3-phenyliminio-3H-phenoxazine mediator is adapted to be used withelectrochemical test sensors. The electrochemical test sensors areadapted to receive a fluid sample and be analyzed using an instrument ormeter. The test sensor assists in determining information related to theanalytes such as analyte concentrations. Analytes that may be measuredinclude glucose, cholesterol, lipid profiles, microalbumin, urea,creatinine, creatine, fructose, lactate, or bilirubin. It iscontemplated that other analyte concentrations may be determined. Theanalytes may be in, for example, a whole blood sample, a blood serumsample, a blood plasma sample, other body fluids like ISF (interstitialfluid) and urine, and non-body fluids.

The test sensors described herein are electrochemical test sensors.Meters used with the electrochemical test sensors may have opticalaspects so as to detect the calibration information and electrochemicalaspects to determine the information related to the analyte (e.g.,analyte concentration of the fluid sample). One non-limiting example ofan electrochemical test sensor is shown in FIG. 1 a. FIG. 1 a depicts atest sensor 10 including a base 11, a capillary channel, and a pluralityof electrodes 16 and 18. A region 12 shows an area that defines thecapillary channel (e.g., after a lid is placed over the base 11). Theplurality of electrodes includes a counter electrode 16 and a working(measuring) electrode 18. The electrochemical test sensor may alsocontain at least three electrodes, such as a working electrode, acounter electrode, a trigger electrode, or a hematocrit electrode. Theworking electrode employed in electrochemical sensors according to theembodiments of the present invention may vary, with suitable electrodesincluding, but not limited to, carbon, platinum, palladium, gold,ruthenium, rhodium and combinations thereof.

The electrodes 16, 18 are coupled to a plurality of conductive leads 15a,b, which, in the illustrated embodiment, terminates with larger areasdesignated as test-sensor contacts 14 a,b. The capillary channel isgenerally located in a fluid-receiving area 19. It is contemplated thatother electrochemical test sensors may be employed with the mediators ofthe present invention.

The fluid-receiving area 19 includes at least one reagent for convertingthe analyte of interest (e.g., glucose) in the fluid sample (e.g.,blood) into a chemical species that is electrochemically measurable, interms of the electrical current it produces, by the components of theelectrode pattern. The reagent typically includes an analyte-specificenzyme that reacts with the analyte and with an electron acceptor toproduce an electrochemically measurable species that may be detected bythe electrodes. The reagent includes a mediator that assists intransferring electrons between the analyte and the electrodes. Thereagent may include binders that hold the enzyme and mediator together,other inert ingredients, or combinations thereof.

A fluid sample (e.g., blood) may be applied to the fluid-receiving area19. The fluid sample reacts with the at least one reagent. Afterreacting with the reagent and in conjunction with the plurality ofelectrodes, the fluid sample produces electrical signals that assist indetermining the analyte concentration. The conductive leads 15 a,b carrythe electrical signal back toward a second opposing end 42 of the testsensor 10 where the test-sensor contacts 14 a,b transfer the electricalsignals into the meter.

Referring to FIG. 1 b, a side view of the test sensor 10 of FIG. 1 a isshown. As shown in FIG. 1 b, the test sensor 10 of FIG. 1 b furtherincludes a lid 20 and a spacer 22. The base 11, the lid 20, and thespacer 22 may be made from a variety of materials such as polymericmaterials. Non-limiting examples of polymeric materials that may be usedto form the base 11, the lid 20, and the spacer 22 includepolycarbonate, polyethylene terephthalate (PET), polyethylenenaphthalate (PEN), polyimide, and combinations thereof. It iscontemplated that other materials may be used in forming the base 11,lid 20, and/or spacer 22.

To form the test sensor 10 of FIGS. 1 a, 1 b, the base 11, the spacer22, and the lid 20 are attached by, for example, an adhesive or heatsealing. When the base 11, the lid 20, and the spacer 22 are attached,the fluid-receiving area 19 is formed. The fluid-receiving area 19provides a flow path for introducing the fluid sample into the testsensor 10. The fluid-receiving area 19 is formed at a first end ortesting end 40 of the test sensor 10. Test sensors of the embodiments ofthe present invention may be formed with a base and a lid in the absenceof a spacer, where the fluid-receiving area is formed directly in thebase and/or the lid.

It is also contemplated that the electrochemical test sensor may beformed in the absence of a spacer. For example, the electrochemical testsensor may include a base and a lid such that a channel (e.g., capillarychannel) is formed when the base and the lid are attached to each other.

The base, spacer and lid may be made from a variety of materials such aspolymeric materials. Non-limiting examples of polymeric materials thatmay be used to form the base, spacer and lid include polycarbonate,polyethylene terephthalate (PET), polystyrene, polyimide, andcombinations thereof. It is contemplated that the base, spacer and lidmay be independently made of other materials. The electrode pattern maybe made from a variety of conductive materials including, but notlimited to, gold, platinum, rhodium, palladium, ruthenium, carbon orcombinations thereof.

In another embodiment, the 3-phenylimino-3H-phenothiazine mediator or3-phenyliminio-3H-phenoxazine mediator may be used in an optical testsensor. The 3-phenylimino-3H-phenothiazine mediator or3-phenyliminio-3H-phenoxazine mediator would be a stable mediator insuch a system.

In one method, a 3-phenylimino-3H-phenothiazine mediator is formed andincludes providing a first reactant including phenothiazine. A firstsolvent is provided in which the phenothiazine has a desired solubilitytherein. A second reactant is provided to assist in forming the3-phenylimino-3H-phenothiazine mediator. A second solvent is provided inwhich the second reactant has a desired solubility therein. The firstreactant and the first solvent are combined together to form a firstreactant solution. The second reactant and second solvent are combinedtogether to form a second reactant solution. The first and secondreactant solutions are combined together to form a reactants solution. Asolution of sodium persulfate is prepared and added to the reactantssolution. The solution of sodium persulfate is typically formed usingthe second solvent (same solvent as used in forming the second reactantsolution). The sodium persulfate causes coupling of the first and secondreactants resulting in a reaction solution with formed product.

In this method, further processing occurs to the reaction solution so asto isolate a 3-phenylimino-3H-phenothiazine mediator. In one embodiment,the 3-phenylimino-3H-phenothiazine mediator is in the form of a salt. Inanother embodiment, the 3-phenylimino-3H-phenothiazine mediator is inthe form of an acid. Some 3-phenylimino-3H-phenothiazine mediators maynot be in the form of the salt or acid.

A second reactant is selected to form the desired3-phenylimino-3H-phenothiazine mediator. For example, the second reagentmay be aniline 2,5-disulfonic acid. When aniline 2,5-disulfonic acid isused, the specific 3-phenylimino-3H-phenothiazine mediator formed is(3-(2′,5′-disulfophenylimino)-3H-phenothiazine mediator.

It is contemplated that other second reactant may be used to formdifferent 3-phenylimino-3H-phenothiazine mediators. For example, thesecond reactant for forming a 3-phenylimino-3H-phenothiazine mediatormay be selected from the following: 4-diethylaminoaniline;4-chloroaniline; 4-ethylaniline; 4-trifluoromethylaniline; methyl4-aminobenzoate; 4-nitroaniline; 4-methoxyaniline;4-(4′-aminophenyl)butyric acid; 4-aminobenzyl amine;4-(2′aminoethyl)aniline; 5-amino-1,3-benzenedicarboxylic acid;4-aminobenzonic acid; 2,5-(4′-aminophenyl)-1,3,4-oxadiazole;4-[2′-(2′-ethanoloxy)ethoxy]ethoxyaniline; and 2,5-disulfoaniline. It iscontemplated that other second reactants may be used to form other3-phenylimino-3H-phenothiazine mediators.

A first solvent is selected that is compatible with the first reactant.It is desirable for the first reactant to have a generally highsolubility into the first solvent. In one method, the first solvent istetrahydrofuran (THF). The first solvent is desirably tetrahydrofuran(THF) because the phenothiazine has a generally high solubility therein.The first solvent is also desirably miscible with the second solvent soas to form a generally or substantially uniform solution.

It is contemplated that other first solvents may be used instead oftetrahydrofuran (THF) such as, for example, N,N-dimethylformamide,methanol, ethanol, 1,4-dioxane and sulfolane. It is also contemplatedthat other first solvents may be used.

A second solvent is selected that is compatible with the secondreactant. It is desirable for the second reactant to have a generallyhigh solubility into the second solvent. In one method, the secondsolvent is water. In another method, the second solvent is a combinationof water and sodium hydroxide (NaOH). The sodium hydroxide is desirablebecause the solubility of at least some second reactants are improved bybeing more basic. It is contemplated that other basic solutions may beadded with the second solvent to achieve improved solubility of thesecond reactant therein. It is contemplated that other second solventsmay be used instead of water.

Sodium persulfate promotes coupling between the first and secondreactants. Sodium persulfate is a desirable coupling agent because it isbelieved to avoid forming undesirable by-products. Using sodiumpersulfate as the coupling agent assists in obtaining a consistent lowbackground current, which means a generally low amount of undesirableby-products are being formed and remaining in the solution.Additionally, the use of sodium persulfate assists in easier isolationof the desired 3-phenylimino-3H-phenothiazine mediator from the reactionby facilitating precipitation of organic material.

To form the 3-phenylimino-3H-phenothiazine mediator, further processingoccurs after the coupling agent is added to the reactants solutionincluding the first reactant, first solvent, second reactant and thesecond solvent. The first solvent (e.g., tetrahydrofuran) may be removedor extracted from the solution. The first solvent may be removed by, forexample, ethyl acetate. Ethyl acetate assists in extracting the firstsolvent and also may assist in removing other undesirable residualorganic material (e.g., water-soluble organic materials) from thereaction solution.

It is contemplated that other compounds may be used to remove the firstsolvent such as, for example, diethyl ether, chloroform anddichloromethane.

The second solvent (e.g., water) is removed from the product by coolingand filtration. By removing the second solvent, this also aids inpreventing or inhibiting decomposition. By preventing or inhibitingdecomposition, the background current will typically be at a moredesired lower level. Residual second solvent (e.g., residual water) notremoved by, for example, cooling and filtration may be removed from theproduct by several methods. For example, the residual second solvent maybe removed by (a) drying in a vacuum oven, (b) adding a compound to theproduct, or (c) lypholization of a solution of the product.

In one process, acetronitrile is added to the residual second solvent toassist in removing the residual second solvent from the solution. It iscontemplated that other compounds may be used to remove the residualsecond solvent such as, for example, acetone and toluene.

It is contemplated that other processing may occur in forming the3-phenylimino-3H-phenothiazine mediator. For example, a processing actbefore the removal of the second solvent may include reconstituting themediator in water, cooling and then filtering at room temperature toremove some of the excess salts. It is also contemplated that otherprocessing acts may occur.

In another method, a 3-phenylimino-3H-phenoxazine mediator is formed andincludes providing a first reactant including phenoxazine. A firstsolvent is provided in which the phenoxazine has a desired solubilitytherein. A second reactant is provided to assist in forming the3-phenylimino-3H-phenoxazine mediator. A second solvent is provided inwhich the second reactant has a desired solubility therein. The firstreactant and the first solvent are combined together to form a firstreactant solution. The second reactant and second solvent are combinedtogether to form a second reactant solution. The first and secondreactant solutions are combined together to form a reactants solution. Asolution of sodium persulfate is prepared and added to the reactantssolution. The solution of sodium persulfate is typically formed usingthe second solvent (same solvent as used in forming the second reactantsolution). The sodium persulfate causes coupling of the first and secondreactants resulting in a reaction solution with formed product.

In this method, further processing occurs to the reaction solution so asto isolate a 3-phenylimino-3H-phenoxazine mediator. In one embodiment,the 3-phenylimino-3H-phenoxazine mediator is in the form of a salt. Inanother embodiment, the 3-phenylimino-3H-phenoxazine mediator is in theform of an acid. Some 3-phenylimino-3H-phenoxazine mediators may not bein the form of a salt or acid.

In this method if forming the 3-phenylimino-3H-phenoxazine mediator, thesame or similar second reactants, first solvents, second solvents may beused as described above with respect to the method of forming the3-phenylimino-3H-phenothiazine mediator. Additionally, the processing ofisolating the 3-phenylimino-3H-phenoxazine mediator by substantiallyremoving at least the first and second solvent may be performed in asimilar or the same manner as described above with respect to3-phenylimino-3H-phenothiazine mediator.

It is contemplated that many different 3-phenylimino-3H-phenothiazinemediators or 3-phenyliminio-3H-phenoxazine mediators may be formed usingthe inventive processes. One desirable example of a phenothiazine thathas been prepared and found to have suitable properties as an NADHmediator is 3-(2′,5′disulfophenylimino)-3H-phenothiazine mediator.Another desirable example is3-(3′,5′-dicarboxy-phenylimino)-3H-phenothiazine mediator that has beenprepared and found to have suitable properties as an NADH mediator.

Among those phenothiazines and phenoxazines that have been prepared andfound to have suitable properties as NADH mediators are3-(4′-chloro-phenylimino)-3H-phenothiazine;3-(4′-diethylamino-phenylimino)-3H-phenothiazine;3-(4′-ethyl-phenylimino)-3H-phenothiazine;3-(4′-trifluoromethyl-phenylimino)-3H-phenothiazine;3-(4′-methoxycarbonyl-phenylimino)-3H-phenothiazine;3-(4′-nitro-phenylimino-3H-phenothiazine;3-(4′-methoxy-phenylimino)-3H-phenothiazine;7-acetyl-3-(4′-methoxycarbonylphenylimino)-3H-phenothiazine;7-trifluoromethyl-3-(4′-methoxycarbonyl-phenylimino)-3H-phenothiazine;3-(4′-ω-carboxy-n-butyl-phenylimino)-3H-phenothiazine;3-(4′-aminomethyl-phenylimino)-3H-phenothiazine; 3-(4′-(2″-(5″-(p-aminophenyl)-1,3,4-oxadiazoyl)phenylimino)-3H-phenothiazine;3-(4′-β-aminoethyl-phenylimino)-3H-phenothiazine;6-(4′-ethylphenyl)amino-3-(4′-ethylphenylimino)-3H-phenothiazine;6-(4′-[2-(2-ethanoloxy)ethoxy]-ethoxyphenyl)amino-3-(4′-[2-(2-ethanoloxy)ethoxy]ethoxyphenylimino)-3H-phenothiazine;3-(4′-[2-(2-ethanoloxy)ethoxy]ethoxy-phenylimino)-3H-phenothiazine;3-(4′-phenylimino)-3H-phenothiazineboronic acid,3-(3′,5′-dicarboxy-phenylimino)-3H-phenothiazine;3-(4′-carboxyphenylimino)-3H-phenothiazine;3-(3′,5-dicarboxy-phenylimino)-3H-phenoxazine;3-(2′,5′-phenylimino)-3H-phenothiazinedisulfonic acid; and3-(3′-phenylimino)-3H-phenothiazinesulfonic acid.

It is contemplated that the phenothiazines and phenoxazines that havebeen prepared and found to have suitable properties may be used withflavoproteins such as FAD-glucose oxidase, flavin-hexose oxidase andFAD-glucose dehydrogenase. It is also contemplated that thephenothiazines and phenoxazines may be prepared to be used and havesuitable properties with quionoproteins such as, for example,PQQ-glucose dehydrogenase.

In another process, the stabilization of 3-phenylimino-3H-phenothiazinemediator or 3-phenylimino-3H-phenoxazine mediator may also be improvedby neutralization or buffering. The neutralization of buffering actassists in stabilizing the mediator so that it is robust during storageconditions that are encountered. It is contemplated that theneutralization or buffering act may occur before or after furtherprocessing has occurred to isolate the mediator. For example, theneutralization or buffering act may occur before the mediator is driedto a powder form. In another example, the neutralization or bufferingact may occur after the mediator has been dried to a powder form.

The neutralizing or buffering agent may be selected from materialsincluding, but not limited to, sodium hydroxide, sodium bicarbonate,sodium phosphate, tetrabutylammonium hydroxide, calcium hydroxide,potassium hydroxide, potassium phosphate, potassium bicarbonate andcombinations thereof. It is contemplated that other materials may beused as the neutralizing or buffering agent.

After the neutralizing or buffering agent is added to the mediatorsolution, the pH is generally from about 5 to about 8. More typically,after the neutralizing or buffering agent is added to the mediatorsolution, the pH is from about 5.5 to about 7 and, even more desirablyfrom about 6 to about 7.

EXAMPLES Example 1 Preparation of(3-(2′,5′-Disulfophenylimino)-3H-Phenothiazine Mediator

Phenothiazine (1.53 mole, 1.1 equivalent, 306 g) was dissolved withstirring into 6.0 L of tetrahydrofuran (THF) and then cooled to 0° C.Aniline 2,5-disulfonic acid (1.38 mole, 350 g) was dissolved in 7.0 L ofwater and 1 M sodium hydroxide (NaOH) (128 ml) was added duringstirring. The aniline 2,5-disulfonic acid solution was added slowly,over the course of about 2 hrs, to the phenothiazine solution, to give awhite, cloudy suspension. The phenothiazine/aniline suspension was at atemperature of about 0° C.-4° C. Sodium persulfate (5.52 mole, 4equivalent, 1314 g) was dissolved in 4.0 L of water to form a sodiumpersulfate solution.

The sodium persulfate solution was added dropwise over 3 hours to thephenothiazine/aniline suspension at a temperature between about 0° C.-3°C. and resulted in a very dark solution. The very dark solution remainedcold using an ice bath and was stirred overnight. The contents were thentransferred to a Buchi rotary evaporator and the tetrahydrofuran wasremoved over the course of about 2 hours at a temperature less than 35°C. After the evaporation act, the remaining solution was transferred toa 25L separator and backwashed with ethyl acetate. The remainingsolution was backwashed 3 times using 2 L of ethyl acetate each time.The reaction fluids were cooled while stirring to −3° C. in anacetone/CO₂ bath. The precipitated solid was filtered through two clothson two 24 cm Buchner funnels on the same day. The precipitated solid wasleft overnight in the funnels to dry and then transferred to a flaskcontaining 2 L of acetonitrile and stirred for about 1 hour at roomtemperature. To remove the residual water, the sample was then filteredand washed with more acetonitrile. The mediator was dried to a constantweight in a vacuum oven at 35° C.

The mediator formed using this process was3-(2′,5′-phenylimino)-3H-phenathiazinedisulfonic acid or3-(2′,5′-disulfophenylimino)-3H-phenothiazine. The mediator is shown asfollows:

Example 2

Background Current of Inventive and Comparative Processes

The background currents of 3-(2′,5′-disulfophenylimino)-3H-phenothiazinemediators prepared by two different processes were compared. TheInventive process for forming the3-(2′,5′-disulfophenylimino)-3H-phenothiazine mediator used sodiumpersulfate as the coupling agent and was substantially the same as theprocess described above in Example 1. This mediator will be referred toas the Inventive mediator. The Comparative process for forming the3-(2′,5′-disulfophenylimino)-3H-phenothiazine mediator used ammoniumpersulfate as the coupling agent. The Comparative process was thesubstantially the same as the Inventive process except for the use ofsodium persulfate as the coupling agent. This mediator will be referredto as the Comparative mediator.

Each of the Inventive and Comparative mediators were separately added toa buffered solution. Each of the buffered solutions included 100 mM ofsodium phosphate. After the Inventive and Comparative mediators wereadded to the buffered solutions, a pH in both solutions was adjusted to7.2. The Inventive and Comparative mediator solutions were thenindividually placed on carbon electrodes. After three seconds, apotential of 250 mV was then applied for five seconds to the carbonelectrodes and then readings of the respective mediator backgroundcurrents were taken.

Referring to FIG. 2, background currents (in nA) of the3-(2′,5′-disulfophenylimino)-3H-phenothiazine mediators were plotted fordifferent lots of mediators formed by the Inventive and Comparativeprocesses. Specifically, five different Comparative mediators (referredto as Comparative mediators 1-5) and four different Inventive mediators(referred to as Inventive mediators 1-4) were tested from differentlots.

As shown in FIG. 2, there were three lots of Comparative mediators thathad very high background currents. See Comparative mediators 1, 4 and 5of FIG. 2 having respective background currents of 2687, 1158 and 1971nA. Comparative mediator 2 had a background current of 75 nA, whileComparative mediator 3 had a background current of 221 nA. All of theInventive mediators 1-4 had a desirable background current of less thanabout 100 nA. Specifically, Inventive mediators 1-4 had respectivebackground currents of 88, 93, 106 and 99 nA.

Example 3 Comparison of Stabilities of3-(2′,5′-Disulfophenylimino)-3H-Phenothiazine Using Different Processes

The stabilities of 3-(2′,5′-disulfophenylimino)-3H-phenothiazineprepared by two different process were compared. The Inventive processfor forming the 3-(2′,5′-disulfophenylimino)-3H-phenothiazine mediatorused sodium persulfate as the coupling agent and was substantially thesame as the process described above in Example 1. This mediator will bereferred to as the Inventive mediator. The Comparative process forforming the 3-(2′,5′-disulfophenylimino)-3H-phenothiazine mediator usedammonium persulfate as the coupling agent. The Comparative process wassubstantially the same as the Inventive process except for the use ofsodium persulfate as the coupling agent. This mediator will be referredto as the Comparative mediator.

The stabilities of the Inventive and Comparative mediators werecompared. Mediators from both the Inventive and Comparative processeswere formulated into respective reagent mixtures. The reagent mixturesfurther included phosphate buffer, Fad-GDH, cellulose polymer andsurfactant. The reagent mixtures were placed onto gold electrodes toform a glucose test sensor. The test sensor samples with Inventive andComparative mediators were exposed to a temperature of −20° C. for aduration of two weeks.

Test sensors formulated with mediators from the same lot of theInventive and Comparative processes were also exposed to a temperatureof 50° C. for a duration of two weeks.

The reagent mixtures included the exposed Inventive mediator orComparative mediator. The response of the electrodes were measured at250 mV applied potential using four different concentrations (0 mg/dl,50 mg/dl, 100 mg/dl and 400 mg/dl) of whole blood glucose samples with aYellow Springs Glucose Analyzer (YSI, Inc., Yellow Springs, Ohio). Theelectrical responses were converted into glucose concentrations usingthe slope and intercept of the respective reagents as referenced to theYSI glucose measurements. The glucose concentrations were tested andcompared for the reagents including the Inventive or Comparativemediator exposed between the temperatures of −20° C. and 50° C. andcompared to see if there was any variance or bias therebetween. Forexample, using 50 mg/dL of glucose, the reagent including the Inventivemediator was compared between the temperatures of −20° C. and 50° C. tosee if there was any variance between the readings. The % bias betweenthese readings was determined.

The % bias is shown for each of the different glucose concentrations andthe Inventive and Comparative mediators are in Table 1 as follows:

TABLE 1 Inventive Process Comparative Process  0 mg/dL −0.7% 7.1%  50mg/dl −1.8% 5.9% 100 mg/dl 0.2% 4.9% 400 mg/dl 2.4% −8.2%

Thus, as shown in Table 1, the3-(2′,5′-disulfophenylimino)-3H-phenothiazine formed using the Inventiveprocess had much greater stability after being exposed to 50° C. for twoweeks than the 3-(2′,5′-disulfophenylimino)-3H-phenothiazine formedusing the Comparative Process. The Inventive process had greaterstability because the measured glucose concentrations did not vary muchafter exposure to the temperature of 50° C. as shown by the low %biases. The Comparative process, on the other hand, had much lessstability because the measured glucose concentrations varied much morethan the Inventive Process after exposure to the temperature of 50° C.as shown by the higher % biases.

Example 4 Effect of Neutralization or Buffering on3-(2′,5′-Disulfophenylimino)-3H-Phenothiazine with Respect to Stability

In each of the neutralization or buffering tests of Example 4, the same3-(2′,5′-disulfophenylimino)-3H-phenothiazine mediator was used. Thesame mediator was also used in the tests that did not include aneutralization or buffering test. The3-(2′,5′-disulfophenylimino)-3H-phenothiazine mediator was formed usingsodium persulfate as the coupling agent and was substantially the sameas the process described above in Example 1.

Example 4 tested three processes using different neutralization orbuffering agents and compared these to two processes that did notinclude a neutralizing or buffering agent. Referring to FIGS. 3 a, 3 b,the processes for forming Mediators 1 and 2 did not include anyneutralization or buffering acts. The process for forming Mediator 1included drying the mediator by a vacuum oven. The process for formingMediator 2 included lyophilizing that was controlled at a pH of 2.4.

Each of the processes of forming Mediators 3-5 included a neutralizationor buffering act. Each of the neutralizing or buffering acts resulted ina pH of 6.1. Specifically, Mediator 3 used 20 mM of sodium phosphate.This solution was formed by taking 5 grams of3-(2′,5′-disulfophenylimino)-3H-phenothiazine mediator and dissolvingthe same into 20 mM sodium phosphate buffer having a pH of 7.2. The pHwas adjusted to 6.1 with 1M NaOH. The use of sodium phosphate buffer isgenerally referred to as a pH adjustment.

Mediator 4 used 1M of sodium hydroxide. This solution was formed bytaking 5 grams of 3-(2′,5′-disulfophenylimino)-3H-phenothiazine anddissolving the same into 100 mL of cold water. 1M sodium hydroxidesolution was added dropwise while stirring until a measured pH of 6.1was obtained. The use of sodium hydroxide in this method neutralizes thesolution and, thus, would be referred to as a neutralizing agent.

Mediator 5 used 1M of sodium bicarbonate. This solution was formed bytaking 5 grams of 3-(2′,5′-disulfophenylimino)-3H-phenothiazine anddissolving the same into 100 mL of cold water. 1M sodium bicarbonatesolution was added dropwise while stirring until a measured pH of 6.1was obtained. The use of sodium bicarbonate in this method neutralizesthe solution and, thus, would be referred to as a neutralizing agent.

Each of the Mediators 3-5 were then froze in an isopropanol/dry ice bathand lyophilized to a dry powder using a VirTis® Model No. 4KBTXLbenchtop 4K Freeze Dryer model (Gardiner, N.Y.).

The dried powder form of the Mediators 1-5 was stressed for two weeksunder various storage conditions. Specifically, nine differentconditions were tested in which the temperatures ranged from −40° C. to50° C. Before being exposed to the temperature conditions, the driedsamples were placed into glass vials, sealed with caps and then stored.Two tests were performed at −40° C. and 30° C. in which a “use”component was added. Specifically, the “use” component included exposingMediators 1-5 to ambient temperature for a period of 30 minutes beforesealing the cap and opening the cap after one week and re-exposing toambient temperature for another period of 30 minutes. This “use”exposure was done only at respective temperatures −40° C. and 30° C. The“initial” testing performed the testing with no storage conditions.

Each of the mediator samples was tested for background current using abackground current screening assay. The mediator samples were preparedas in Example 1 and with a pH adjustment to 7.2 using 100 mM sodiumphosphate as described in Example 2. These mediator samples were addedto a carbon electrode. After three seconds, a 250 mV potential wasapplied for a period of five seconds and then the background current wasmeasured.

As shown in FIG. 3 a, the background current (in nA) was much lower inthe mediators that included the neutralization or buffering act whenexposed to higher temperatures during this time period. Comparebackground currents at temperatures greater than 25° C. for Mediators1-5. This was especially the case at the highest exposure temperature of50° C.

FIG. 3 b depicts the change in the background current (%) between themeasured background current of FIG. 3 a from the baseline that wasmeasured before the different exposures. Similarly, the backgroundcurrent (%) was much lower in the mediators that included theneutralization or buffering act when exposed to higher temperaturesduring this time period. Compare % change in background currents attemperatures greater than 25° C. for Mediators 1-5. This was especiallythe case at the highest exposure temperature of 50° C.

Process A

A method of forming a 3-phenylimino-3H-phenothiazine mediator, themethod comprising the acts of:

providing a first reactant including phenothiazine;

providing a first solvent in which the phenothiazine has a desiredsolubility therein;

providing a second reactant to assist in forming the3-phenylimino-3H-phenothiazine mediator;

providing a second solvent in which the second reactant has a desiredsolubility therein;

combining the first reactant, first solvent, second reactant and secondsolvent to form a reactants solution;

adding sodium persulfate to the reactants solution to couple the firstand second reactants resulting in a reaction solution including the3-phenylimino-3H-phenothiazine mediator; and

after adding the sodium persulfate, further processing to the reactionsolution including the 3-phenylimino-3H-phenothiazine mediator so as toisolate the 3-phenylimino-3H-phenothiazine mediator.

Process B

The method of alternative process A wherein the first solvent includestetrahydrofuran (THF).

Process C

The method of alternative process A wherein the second solvent includeswater.

Process D

The method of alternative process C wherein the second solvent furtherincludes sodium hydroxide.

Process E

The method of alternative process A wherein combining the firstreactant, first solvent, second reactant and second solvent includingthe acts of combining the first reactant and the first solvent to form afirst reactant solution, and combining the second reactant and thesecond solvent to form a second reactant solution before the firstreactant, first solvent, second reactant and second solvent are combinedtogether to form the reactants solution.

Process F

The method of alternative process A wherein further processing includesgenerally removing the second solvent by adding acetronitrile.

Process G

The method of alternative process A wherein further processing includesgenerally removing the first solvent by adding ethyl acetate.

Process H

The method of alternative process A wherein the second reactant includesaniline 2,5-disulfonic acid.

Process I

The method of alternative process A wherein the further processingincludes substantially removing at least the first solvent and secondsolvent from the second solution so as to isolate the3-phenylimino-3H-phenothiazine mediator.

Process J

The method of alternative process A wherein the3-phenylimino-3H-phenothiazine mediator is in the form of a salt.

Process K

The method of alternative process A wherein the3-phenylimino-3H-phenothiazine mediator is in the form of an acid.

Process L

A method of forming a 3-phenylimino-3H-phenoxazine mediator, the methodcomprising the acts of:

providing a first reactant including phenoxazine;

providing a first solvent in which the phenoxazine has a desiredsolubility therein;

providing a second reactant to assist in forming the3-phenylimino-3H-phenoxazine mediator;

providing a second solvent in which the second reactant has a desiredsolubility therein;

combining the first reactant, first solvent, second reactant and secondsolvent to form a reactants solution;

adding sodium persulfate to the reactants solution to couple the firstand second reactants resulting in a reaction solution including the3-phenylimino-3H-phenoxazine mediator; and

after adding the sodium persulfate, further processing to the reactionsolution including the 3-phenylimino-3H-phenoxazine mediator so as toisolate the 3-phenylimino-3H-phenoxazine mediator.

Process M

The method of alternative process L wherein the first solvent includestetrahydrofuran (THF).

Process N

The method of alternative process L wherein the second solvent includeswater.

Process O

The method of alternative process N wherein the second solvent furtherincludes sodium hydroxide.

Process P

The method of alternative process L wherein combining the firstreactant, first solvent, second reactant and second solvent includingthe acts of combining the first reactant and the first solvent to form afirst reactant solution, and combining the second reactant and thesecond solvent to form a second reactant solution before the firstreactant, first solvent, second reactant and second solvent are combinedtogether to form the reactants solution.

Process Q

The method of alternative process L wherein further processing includesgenerally removing residual second solvent by adding acetronitrile.

Process R

The method of alternative process L wherein further processing includesgenerally removing the first solvent by adding ethyl acetate.

Process S

The method of alternative process L wherein the further processingincludes substantially removing at least the first solvent and secondsolvent from the second solution so as to isolate the3-phenylimino-3H-phenoxazine mediator.

Process T

The method of alternative process L wherein the3-phenylimino-3H-phenoxazine mediator is in the form of a salt.

Process U

The method of alternative process L wherein the3-phenylimino-3H-phenoxazine mediator is in the form of an acid.

Process V

A method of forming and stabilizing a 3-phenylimino-3H-phenothiazinemediator or 3-phenylimino-3H-phenoxazine mediator, the method comprisingthe acts of:

providing a first reactant including phenothiazine or phenoxazine;

providing a first solvent in which the phenothiazine or the phenoxazinehas a desired solubility therein;

providing a second reactant to assist in forming the3-phenylimino-3H-phenothiazine mediator or the3-phenylimino-3H-phenothiazine mediator;

providing a second solvent in which the second reactant has a desiredsolubility therein;

combining the first reactant, first solvent, second reactant and secondsolvent to form a reactants solution;

adding a coupling agent to the reactants solution to couple the firstand second reactants resulting in a reaction solution including the3-phenylimino-3H-phenothiazine mediator or the3-phenylimino-3H-phenoxazine mediator; and

after adding the coupling agent, further processing to the reactionsolution including the 3-phenylimino-3H-phenothiazine mediator or the3-phenylimino-3H-phenoxazine mediator so as to isolate the3-phenylimino-3H-phenothiazine mediator or the3-phenylimino-3H-phenoxazine mediator; and

stabilizing the 3-phenylimino-3H-phenothiazine mediator or the3-phenylimino-3H-phenoxazine mediator to a pH of from about 5 to about8.

Process W

The method of alternative process V wherein the pH is from about 5.5 toabout 7.

Process X

The method of alternative process W wherein the pH is from about 6 toabout 7.

Process Y

The method of alternative process W wherein the stabilizing the3-phenylimino-3H-phenothiazine mediator or the3-phenylimino-3H-phenoxazine mediator includes adding sodium hydroxide,sodium bicarbonate, sodium phosphate, tetrabutylammonium hydroxide,calcium hydroxide, potassium hydroxide, potassium phosphate, potassiumbicarbonate or combinations thereof.

While the present invention has been described with reference to one ormore particular embodiments, those skilled in the art will recognizethat many changes may be made thereto without departing from the spiritand scope of the present invention. Each of these embodiments, andobvious variations thereof, is contemplated as falling within the spiritand scope of the invention.

1-25. (canceled)
 26. A 3-phenylimino-3H-phenothiazine mediator formed bythe process comprising the acts of: providing a first reactant includingphenothiazine; providing a first solvent in which the phenothiazine hasa desired solubility therein; providing a second reactant to assist informing the 3-phenylimino-3H-phenothiazine mediator; providing a secondsolvent in which the second reactant has a desired solubility therein;combining the first reactant, first solvent, second reactant and secondsolvent to form a reactants solution; adding sodium persulfate to thereactants solution to couple the first and second reactants resulting ina reaction solution including the 3-phenylimino-3H-phenothiazinemediator; and after adding the sodium persulfate, further processing tothe reaction solution including the 3-phenylimino-3H-phenothiazinemediator so as to isolate the 3-phenylimino-3H-phenothiazine mediator.27. The mediator of claim 26, wherein the first solvent includestetrahydrofuran (THF).
 28. The mediator of claim 26, wherein the secondsolvent includes water.
 29. The mediator of claim 28, wherein the secondsolvent further includes sodium hydroxide.
 30. The mediator of claim 26wherein combining the first reactant, first solvent, second reactant andsecond solvent including the acts of combining the first reactant andthe first solvent to form a first reactant solution, and combining thesecond reactant and the second solvent to form a second reactantsolution before the first reactant, first solvent, second reactant andsecond solvent are combined together to form the reactants solution. 31.The mediator of claim 26, wherein further processing includes generallyremoving the second solvent by adding acetronitrile.
 32. The mediator ofclaim 26, wherein further processing includes generally removing thefirst solvent by adding ethyl acetate.
 33. The mediator of claim 26,wherein the second reactant includes aniline 2,5-disulfonic acid. 34.The mediator of claim 26, wherein the further processing includessubstantially removing at least the first solvent and second solventfrom the second solution so as to isolate the3-phenylimino-3H-phenothiazine mediator.
 35. The mediator of claim 26,wherein the 3-phenylimino-3H-phenothiazine mediator is in the form of asalt.
 36. The mediator of claim 26, wherein the pH is from about 5 toabout
 8. 37. The mediator of claim 36, wherein the pH is from about 5.5to about
 7. 38. The mediator of claim 37, wherein the pH is from about 6to about
 7. 39. The mediator of claim 36, wherein the pH is from about5.5 to about
 6. 40. The mediator of claim 26, further includes aneutralizing agent.
 41. The mediator of claim 40, wherein theneutralizing agent is sodium hydroxide.
 42. The mediator of claim 40,wherein the neutralizing agent is sodium bicarbonate.
 43. The mediatorof claim 26, further including a buffer.
 44. A3-phenylimino-3H-phenoxazine mediator formed by the process comprisingthe acts of: providing a first reactant including phenoxazine; providinga first solvent in which the phenoxazine has a desired solubilitytherein; providing a second reactant to assist in forming the3-phenylimino-3H-phenoxazine mediator; providing a second solvent inwhich the second reactant has a desired solubility therein; combiningthe first reactant, first solvent, second reactant and second solvent toform a reactants solution; adding sodium persulfate to the reactantssolution to couple the first and second reactants resulting in areaction solution including the 3-phenylimino-3H-phenoxazine mediator;and after adding the sodium persulfate, further processing to thereaction solution including the 3-phenylimino-3H-phenoxazine mediator soas to isolate the 3-phenylimino-3H-phenoxazine mediator.
 45. Themediator of claim 44, wherein the first solvent includes tetrahydrofuran(THF).
 46. The mediator of claim 44, wherein the second solvent includeswater.
 47. The mediator of claim 46, wherein the second solvent furtherincludes sodium hydroxide.
 48. The mediator of claim 44, whereincombining the first reactant, first solvent, second reactant and secondsolvent including the acts of combining the first reactant and the firstsolvent to form a first reactant solution, and combining the secondreactant and the second solvent to form a second reactant solutionbefore the first reactant, first solvent, second reactant and secondsolvent are combined together to form the reactants solution.
 49. Themediator of claim 44, wherein further processing includes generallyremoving residual second solvent by adding acetronitrile.
 50. Themediator of claim 44, wherein further processing includes generallyremoving the first solvent by adding ethyl acetate.
 51. The mediator ofclaim 44, wherein the further processing includes substantially removingat least the first solvent and second solvent from the second solutionso as to isolate the 3-phenylimino-3H-phenoxazine mediator.
 52. Themediator of claim 44, wherein the 3-phenylimino-3H-phenoxazine mediatoris in the form of a salt.
 53. The mediator of claim 44, wherein the3-phenylimino-3H-phenoxazine mediator is in the form of an acid.
 54. Themediator of claim 44, wherein the pH is from about 5 to about
 8. 55. Themediator of claim 54, wherein the pH is from about 5.5 to about
 7. 56.The mediator of claim 55, wherein the pH is from about 6 to about
 7. 57.The mediator of claim 54, wherein the pH is from about 5.5 to about 6.58. The mediator of claim 44, further includes a neutralizing agent. 59.The mediator of claim 58, wherein the neutralizing agent is sodiumhydroxide.
 60. The mediator of claim 58, wherein the neutralizing agentis sodium bicarbonate.
 61. The mediator of claim 44, further including abuffer.